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Research Articles: Neurobiology of Disease

Astrocyte transforming growth factor beta 1 protects synapses against


Aβ oligomers in Alzheimer's disease model
Luan Pereira Diniz1, Vanessa Tortelli1, Isadora Matias1, Juliana Morgado1, Ana Paula Bérgamo Araujo1,
Helen M. Melo2, Gisele S. Seixas da Silva2,5, Soniza V. Alves-Leon3, Jorge M. de Souza3, Sergio T.
Ferreira2,4, Fernanda G. De Felice2,6 and Flávia Carvalho Alcantara Gomes1
1
Instituto de Ciências Biomédicas
2
Instituto de Bioquímica Médica Leopoldo de Meis
3
Hospital Universitário Clementino Fraga Filho
4
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro
5
Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, Rio de Janeiro, RJ; BRASIL
6
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6 Canada.

DOI: 10.1523/JNEUROSCI.3351-16.2017

Received: 25 October 2016

Revised: 28 May 2017

Accepted: 31 May 2017

Published: 8 June 2017

Author contributions: L.P.D., V.T., I.M., F.G.D.F., and F.C.A.G. designed research; L.P.D., V.T., I.M., J.M.,
A.P.B.A., H.M.M., G.S.S.d.S., S.V.A.-L., and J.M.d.S. performed research; L.P.D., V.T., I.M., J.M., H.M.M.,
G.S.S.d.S., S.T.F., F.G.D.F., and F.C.A.G. analyzed data; L.P.D., V.T., S.T.F., F.G.D.F., and F.C.A.G. wrote the
paper; S.V.A.-L. and J.M.d.S. contributed unpublished reagents/analytic tools.

Conflict of Interest: The authors declare no competing financial interests.

We thank Marcelo Meloni and Grasiela Ventura for technical assistance. This work was supported by grants
from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Saúde,
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Carlos Chagas Filho
de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Address correspondence to: Flávia Carvalho Alcantara Gomes, Instituto de Ciências Biomédicas,
Universidade Federal do Rio de Janeiro (UFRJ), 21941-902, Rio de Janeiro, RJ, BRASIL. fgomes@icb.ufrj.br

Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3351-16.2017

Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted
version of this article is published.

Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading
process.

Copyright © 2017 the authors




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